Van de Loo, et al., 1995. An oleate 12-hydroxylase from Ricinus communis is a fatty acyl desaturase homolog. P.N.A.S. 92:6743-6747

This paper is a classic for its pioneering approach, 'high-throughput DNA sequencing' to try to identify  a gene for a specific enzymatic function, where no directly homologous sequences are available. While most of the technical aspects are beyond our course, the key technique is one of differential hybridization, which allows the identification of cDNA clones in a library by comparing their abundance in 2 tissues, ie seed vs leaf.

Questions two guide your reading. We'll discuss on Thurs, Oct 5th so please submit your responses at that time.

1. Given that prior to this, no known sequence data for this enzyme and its gene were known, what are the criteria the authors are using in their search?

2. What is the most direct proof the authors have that they have identified the correct gene? (Are you convinced?)

3. What promoter is being used to drive expression of the cloned gene in transgenic tobacco. Would you expect expression in leaf tissue as well as in seeds?

4. Is the putative new hydroxylase gene expressed in leaves of the transgenic tobacco plants? Do the authors show any data on this?

5. Speculate on why there is such low levels of ricinoleic acid in the transgenic plants.